Usp2a peptides and antibodies

ABSTRACT

The invention relates to novel USP2a peptides and antibodies, as well as nucleic acids related to them. The peptides, antibodies and the nucleic acids are useful for the detection, staging and monitoring of the progression of cancer, as well as for determining or monitoring the efficacy of treatment.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/659,173, filed Oct. 24, 2012, which claims priority to U.S.Provisional Patent Application No. 61/551,567 filed Oct. 26, 2011 whichis incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled20151007USDIV3SEQLST.txt, created on May 16, 2013 which is 12,401 bytesin size. The information in the electronic format of the sequencelisting is incorporated herein by reference in its entirety.

FIELD OF INVENTION

The invention relates to peptides and antibodies havingimmunospecificity for USP2a polypeptides and proteins, as well asnucleic acids related to these peptides and antibodies, and methods forusing these peptides and antibodies.

BACKGROUND OF THE INVENTION

According to the American Cancer Society, in 2009 there were over190,000 cases of prostate cancer reported and over 27,000 related deathsin the United States.

The ubiquitin-specific protease 2a (USP2a) can deubiquitinate theantiapoptotic proteins fatty acid synthase (FAS) and Mdm2. It has beenshown that when USP2a is also overexpressed in nontransformed cells, itexhibits oncogenic behavior both in vitro and in vivo and also preventsapoptosis induced by chemotherapeutic agents. Notably, USP2a silencingin several human cancer cell lines can result in apoptosis.

USP2a is overexpressed in about 50% of human prostate tumors, and itsoncogenicity in prostate cancer as well as its anti-apoptotic role in avariety of human tumor cell lines have been thoroughly demonstrated,making USP2a a good therapeutic target and prognostic marker in humancancer. However, currently available anti-USP2a antibodies are notspecific enough and therefore are likely to cross-react with otherproteins. This lack of specificity renders the current anti-USP2aantibodies less useful in both therapeutic discovery and as a marker inthe analysis of patient tumors. Consequently, there remains a need forbetter USP2a antibodies useful in the diagnosis, stratification andprognosis of disorders and conditions, especially cancers.

SUMMARY OF THE INVENTION

The present invention provides peptides and antibodies havingimmunospecifity for USP2a polypeptides and proteins. The presentinvention further provides nucleic acids related to these peptides andantibodies. The present invention also provides diagnostic andtherapeutic compositions and methods using these peptides, antibodiesand nucleic acids.

In one embodiment, the present invention provides an isolated and/orrecombinant USP2a peptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO. 1, SEQ ID NO. 2 and variantsthereof.

In a further embodiment, the present invention provides antibodiesspecifically immunoreactive with one or more peptides comprising anamino acid sequence selected from the group consisting of SEQ ID NO. 1,SEQ ID NO 2, and variants thereof.

In one embodiment, the present invention provides an isolated nucleicacid molecule comprising a sequence encoding a peptide selected from thegroup consisting of SEQ ID NO. 1, SEQ ID NO. 2 and variants thereof.

In a further embodiment, the USP2a peptide, antibody, or nucleic acid ofthe present invention may further comprise a detectable label. The USP2apeptide, antibody, or nucleic acid of the present invention may beimmobilized on a substrate.

In one embodiment, the present invention provides a composition fordetecting USP2a polypeptides or proteins in a sample which comprises aprimary antibody specifically immunoreactive with a USP2a peptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO. 1, SEQ ID NO. 2 and variants thereof. In a furtherembodiment, the primary antibody may be labeled with a detectable label.

In one embodiment, the composition of the present invention may furthercomprise a secondary antibody. In a further embodiment, the secondaryantibody may react with the primary antibody. In another embodiment, thesecondary antibody is specifically immunoreactive with a USP2a peptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO. 1, SEQ ID NO. 2 and variants thereof. In a furtherembodiment, the secondary antibody may be labeled with a detectablelabel.

In one embodiment, the present invention provides a composition fordetecting one or more USP2a polypeptides or proteins in a sample,comprising a nucleic acid which comprises at least a portion of asequence encoding a peptide selected from the group consisting of SEQ IDNO. 1, SEQ ID NO. 2 and variants thereof. In a further embodiment, thenucleic acid preferably comprises at least from about 5 up to about 80nucleic acid bases encoding all or a portion of a peptide selected fromthe group consisting of SEQ ID NO. 1, SEQ ID NO. 2 and variants thereof.In another embodiment, the nucleic acid may be labeled with a detectablelabel.

In one embodiment, the present invention provides a method fordetecting, diagnosing, staging or monitoring the progression of adisease in a subject, where the method comprises: a) obtaining a samplefrom the subject, b) contacting the sample with an antibody specificallyimmunoreactive with a USP2a peptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2 andvariants thereof, c) determining a level of USP2a protein in the sampledetected by said antibody, and d) comparing the level of the USP2aprotein in the sample to a baseline level of the USP2a protein; where adifference in levels of the USP2a protein in the sample as compared to abaseline of greater than 1%, 5%, 50%, 75%, 100% or more is indicative ofthe presence, stage or progression of the disease in the subject. In afurther embodiment the disease may be cancer.

In a further embodiment, the detection of the USP2a protein may beaccomplished using any type of immunoassay, for example, an immunoassayselected from immunoblot assay, enzyme-linked immunosorbant assay(ELISA), radioimmunoassay (RIA), immunoprecipitation,immunohistochemistry (IHC) or immunofluorescence.

In one embodiment, the present invention provides a method fordetecting, diagnosing, staging or monitoring the progression of a USP2adisease or condition in a subject, where the method comprises: a)obtaining a sample from the subject, b) contacting the sample with annucleic specifically immunoreactive with a USP2a nucleic acid comprisingan amino acid sequence selected from the group consisting of SEQ ID NO.1, SEQ ID NO. 2 and variants thereof, c) determining a level of USP2anucleic acid in the sample detected by said nucleic acid, and d)comparing the level of the USP2a nucleic acid in the sample to abaseline level of the USP2a nucleic acid; where a difference in levelsof the USP2a nucleic acid in the sample as compared to a baseline ofgreater than 1%, 5%, 50%, 75%, 100% or more is indicative of thepresence, stage or progression of the USP2a-mediated disease in thesubject.

In a further embodiment, the USP2a nucleic acid may be a DNA or an RNA.In further embodiment, the present invention provides a method furthercomprising determining the efficacy of therapeutic intervention ortreatment by a step of modifying therapeutic intervention or treatmentof the USP2a disease or condition based on the difference in USP2alevels.

In a further embodiment, the detection of USP2a nucleic acid may beaccomplished by a method selected from the group consisting ofpolymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR), insitu hybridization, Southern blot, Northern blot, sequence analysis,gene microarray analysis, and detection of a reporter gene.

In one embodiment, the provided sample is obtained from a patient to bediagnosed, monitored or treated for the disease, and the baseline levelis from a control sample from a patient not having the disease.

In another embodiment, the sample is from a subject who is known to havethe disease, and the baseline level comprises a level of USP2a from aprevious sample from the same subject, wherein a difference in the levelof the USP2a indicates that the subject is at a different disease stage,or is indicative of the efficacy of therapeutic intervention ortreatment, e.g., the responsiveness of the patient to therapy.

In a further embodiment, the sample of the invention may comprise, forexample, a bodily fluid sample, a tissue sample, or a cell sample. In afurther embodiment, the sample may be immobilized on a substrate.

In one embodiment, the method of the present invention is used todetermine the prognosis of the disease in a patient. In anotherembodiment, the method of the present invention is used to determine thesusceptibility of a patient to a therapeutic treatment.

In one embodiment, the present invention provides for an antibodyobtained by the method which comprises the steps of: a) contacting asubject with at least one USP2a peptide comprising a portion of a USP2aprotein, wherein the USP2a peptide is from about 5 to about 30 aminoacids in length and has a sequence selected from the group consisting ofSEQ ID NO. 1, SEQ ID NO. 2 and variants thereof, and b) collecting asample containing the USP2a antibody from the subject. In a furtherembodiment, the sample collected contains antiserum that has beenimmunopurified.

In another embodiment, the antibody created by the method of the presentinvention is a monoclonal antibody. In a further embodiment, theantibody created by the method of the present invention contains adetectable label.

In one embodiment, the present invention provides a method for detectingthe presence or amount of USP2a protein in a sample comprisingcontacting the sample with the isolated antibody of the presentinvention.

In one embodiment, the present invention also provides an expressionvector comprising a nucleic acid which comprises a sequence encoding apeptide selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.2, and variants thereof. In a further embodiment, the present inventionprovides a host cell transfected with the expression vector.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to peptides and antibodies havingimmunospecificity for USP2a peptides and proteins, as well as nucleicacids related to these peptides and antibodies, and methods for usingthese peptides and antibodies.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of methods featured in the invention, suitablemethods and materials are described below.

Definitions

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are provided below. Thedefinitions are not meant to be limiting in nature and serve to providea clearer understanding of certain aspects of the present invention.

The term “peptide” refers to a natural or synthetic molecule comprisingtwo or more amino acids linked by the carboxyl group of one amino acidand the amino group of another. A peptide of the present invention isnot limited by length, thus the term encompasses polypeptide andprotein. A “USP2a peptide” of the present invention is a peptidefragment derived from a USP2a protein and is preferably between about 2to about 100 amino acids in length, more preferably between about 5 toabout 50 amino acids in length, more preferably between about 10 toabout 30 amino acids in length, even more preferably between about 10 toabout 20 amino acids in length. The terms polypeptide and proteinsometimes are used interchangeably. A “USP2a polypeptide” or “USP2aprotein” may refer to an entire USP2a protein (SEQ ID NO. 3), or tofragment or variant thereof. A “fragment,” as used herein, refers to apolypeptide or protein that is a portion of another polypeptide orprotein. For example, fragments of proteins may comprise polypeptidesobtained by digesting full-length protein isolated from cultured cells.As used herein, a “variant” of a peptide or protein is defined as onewhich is at least the functional equivalent of the parent molecule butwhich may differ in sequence by no more than 20% in sequence from theparent molecule.

The term “immunogenic epitope” refers to a portion of a peptide thatelicits an antibody response in an animal, as determined by any methodknown in the art, for example, by the methods for generating antibodiesdescribed herein. (See, for example, Geysen et al., 1983, Proc. Natl.Acad. Sci. USA, 81:3998-4002). The term “antigenic epitope” refers to aportion of a protein to which an antibody can immunospecifically bind toits antigen as determined by any method well known in the art, forexample, by the immunoassays described herein. Antigenic epitopes neednot necessarily be immunogenic. Peptides that function as epitopes maybe produced by any conventional means. (See, e.g., Houghten, 1985, Proc.Natl. Acad. Sci. USA, 82:5131-5135; and as described in U.S. Pat. No.4,631,211).

The term “isolated,” with respect to peptides, nucleic acids, orantibodies, refers to the material that is removed from its originalenvironment (e.g., the natural environment if it is naturally occurringmaterial). For example, a naturally-occurring nucleic acid or peptide orantibody present in a living animal is not isolated, but the samenucleic acid or peptide or antibody, which is separated from some or allof the coexisting materials in the natural system, is isolated. Suchisolated nucleic acid could be part of a vector and such isolatednucleic acid or peptide or antibody could be part of a composition, andstill be isolated in that the vector or composition is not part of itsnatural environment. An “isolated” peptide, nucleic acid or antibody,also includes material synthesized, or produced by recombinant DNAtechnology, as well as preparations such as serum containing an antibodyof the invention.

The term “specifically immunoreactive,” as used herein, refers to ameasurable and reproducible specific immunoreaction such as bindingbetween a peptide and an antibody, that is determinative of the presenceof the peptide in the presence of a heterogeneous population of peptidesand other biologics. The term specifically immunoreactive may includespecific recognition of structural shapes and surface features. Thus,under designated conditions, an antibody specifically immunoreactive toa particular peptide will not significantly bind to other peptidespresent in the sample. An antibody specifically immunoreactive to apeptide has an association constant of at least 10³M⁻¹ or 10⁴M⁻¹,sometimes about 10⁵M⁻¹ or 10⁶M⁻¹, in other instances 10⁶M⁻¹ or 10⁷M⁻¹,preferably about 10⁸M⁻¹ to 10⁹M⁻¹, and more preferably, about 10¹⁰M⁻¹ to10¹¹M⁻¹ or higher. A variety of immunoassay formats can be used todetermine if antibodies are specifically immunoreactive to a particularpeptide. For example, solid-phase ELISA immunoassays are routinely usedto select monoclonal antibodies specifically immunoreactive with apeptide. See, e.g., Harlow and Lane (1988) Antibodies, A LaboratoryManual, Cold Spring Harbor Publications, New York, for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity.

The term “antibody” refers to an immunoglobulin, whether natural orpartially or wholly synthetically produced. All derivatives thereof thatmaintain specific binding ability are also included in the term. Theterm also covers any protein having a binding domain that is homologousor largely homologous to an immunoglobulin binding domain. An antibodymay specifically immunoreactive to a given antigen (e.g., a USP2apeptide of the invention). The term antibody as used herein is intendedto include whole antibodies of any isotype (IgG, IgA, IgM, IgE, etc),and fragments thereof. An antibody of the present invention alsoincludes an antibody preparation, e.g., a serum (antiserum). Antibodiescan be fragmented using conventional techniques and the fragmentsscreened for utility in the same manner as whole antibodies. Thus, theterm includes segments of proteolytically-cleaved orrecombinantly-prepared portions of an antibody molecule that selectivelyreacts with a certain protein or peptide. Non-limiting examples of suchproteolytic and/or recombinant fragments include Fab, F(ab′)2, Fab′, Fv,and single chain antibodies (scFv) containing a V[L] and/or V[H] domainjoined by a peptide linker. The scFv's may be covalently ornon-covalently linked to form antibodies having two or more bindingsites.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variants that mayarise during production of the monoclonal antibody, such variantsgenerally being present in minor amounts. In contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Thistype of antibodies is produced by the daughter cells of a singleantibody-producing hybridoma. A monoclonal antibody typically displays asingle binding affinity for any epitope with which it immunoreacts.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. Monoclonal antibodies recognize onlyone type of antigen The monoclonal antibodies herein include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies. Thepreparation of antibodies, whether monoclonal or polyclonal, is know inthe art. Techniques for the production of antibodies are well known inthe art and described, e.g. in Harlow and Lane “Antibodies, A LaboratoryManual”, Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane“Using Antibodies: A Laboratory Manual” Cold Spring Harbor LaboratoryPress, 1999.

A monoclonal antibody may contain an antibody molecule having aplurality of antibody combining sites, each immunospecific for adifferent epitope, e.g., a bispecific monoclonal antibody. Monoclonalantibodies may be obtained by methods known to those skilled in the art.Kohler and Milstein (1975), Nature, 256:495-497; U.S. Pat. No.4,376,110; Ausubel et al. (1987, 1992), eds., Current Protocols inMolecular Biology, Greene Publishing Assoc. and Wiley Interscience,N.Y.; Harlow and Lane (1988), Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory; Colligan et al. (1992, 1993), eds., CurrentProtocols in Immunology, Greene Publishing Assoc. and WileyInterscience, N.Y.; Iyer et al., Ind. J. Med. Res., (2000), 123:561-564.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides. ESTs, chromosomes,cDNAs, mRNAs, and rRNAs are representative examples of molecules thatmay be referred to as nucleic acids. The term nucleic acid is notlimited by length, thus encompasses “polynucleotide” and“oligonucleotide.” A “nucleic acid encoding a USP2a peptide,” as usedherein, is preferably between about 6 to about 300 nucleotides inlength, more preferably between about 15 to about 150 nucleotides inlength, and more preferably, between about 30 to about 90 nucleotides inlength, and more preferably, between about 30 to about 60 nucleotides inlength

As used herein, the term “stringent conditions” refers to conditionswhere only nucleic acid sequences which are very similar to each otherwill hybridize. The precise conditions determining the stringency of aparticular hybridization include not only the ionic strength,temperature, and the concentration of destabilizing agents such asformamide, but also on factors such as the length of the nucleic acidsequences, their base composition, the percent of mismatched base pairsbetween the two sequences, and the frequency of occurrence of subsets ofthe sequences (e.g., small stretches of repeats) within othernon-identical sequences. The conditions generally refer to hybridizationat either (1) 1×SSC (10×SSC=3 M NaCl, 0.3 M Na₃-citrate.2H₂O (88g/liter), pH to 7.0 with 1 M HCl), 1% SDS (sodium dodecyl sulfate),0.1-2 mg/ml denatured salmon sperm DNA at 65° C.; (2) 1×SSC, 50%formamide, 1% SDS, 0.1-2 mg/ml denatured salmon sperm DNA at 42° C.; (3)1% bovine serum albumen (fraction V), 1 mM Na₂.EDTA, 0.5 M NaHPO₄ (pH7.2) (1 M NaHPO₄=134 g Na₂HPO₄7H₂O, 4 ml 85% H.sub.3PO₄ per liter), 7%SDS, 0.1-2 mg/ml denatured salmon sperm DNA at 65° C.; (4) 50%formamide, 5×SSC, 0.02 M Tris-HCl (pH 7.6), 1×Denhardt's solution(100×=10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serumalbumin (fraction V), water to 500 ml), 10% dextran sulfate, 1% SDS,0.1-2 mg/ml denatured salmon sperm DNA at 42° C.; (5) 5×SSC,5×Denhardt's solution, 1% SDS, 100 [g/ml denatured salmon sperm DNA at65° C.; or (6) 5×SSC, 5×Denhardt's solution, 50% formamide, 1% SDS, 100μg/ml denatured salmon sperm DNA at 42° C., with high stringency washesof either (1) 0.3-0.1×SSC, 0.1% SDS at 65° C.; or (2) 1 mM Na₂EDTA, 40mM NaHPO₄ (pH 7.2), 1% SDS at 65° C. The above conditions are intendedto be used for DNA-DNA hybrids of 50 base pairs or longer. Where thehybrid is believed to be less than 18 base pairs in length, thehybridization and wash temperatures should be 5-10° C. below that of thecalculated T_(m) of the hybrid, where T_(m) in ° C.=(2× the number of Aand T bases)+(4× the number of G and C bases). For hybrids believed tobe about 18 to about 49 base pairs in length, the T_(m) in ° C.=(81.5°C.+16.6(log₁₀ M)+0.41(% G+C)−0.61 (% formamide)−500/L), where “M” is themolarity of monovalent cations (e.g., Na⁺), and “L” is the length of thehybrid in base pairs.

The term “primer” refers to a nucleic acid which binds to a targetnucleic acid of complementary sequence through one or more types ofchemical bonds, usually through complementary base pairing, e.g.,hydrogen bond formation. As used herein, a primer may include natural(i.e., A, G, C, or T) or a modified base (7-deazaguanosine, inosine,etc.) or a sugar moiety. In addition, the bases in a primer may bejoined by a linkage other than a phosphodiester bond, so long as it doesnot interfere with hybridization. Thus, for example, primers may bepeptide nucleic acids in which the constituent bases are joined bypeptide bonds rather than phosphodiester linkages. It will be understoodby one of skill in the art that primers may bind target sequenceslacking complete complementarity with the probe sequence depending uponthe stringency of the hybridization conditions. A “probe” is a primerthat is directly or indirectly labeled with a detectable label bymethods known in the art. The primer is capable of initiating strandelongation. By assaying for the presence, absence, or the level of theprobe binding or primer elongation product, one can detect the presence,absence, or the level of the target nucleic acid. Preferably, the probeor primer of the present invention is between about 8 to 100 nucleotidesin length, more preferably between about 12 to 50 nucleotides, morepreferably between about 12 to 35 nucleotides in length.

As used herein, the term “detectable label” refers to a compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,or chemical means. The detectable label can be selected, e.g., from agroup consisting of radioisotopes, fluorescent compounds, enzymes, andenzyme co-factors.

The phrase “conservative amino acid substitution” as used herein is onein which the amino acid residue is replaced with an amino acid residuehaving a side chain with a similar charge. Families of amino acidresidues having side chains with similar charges have been defined inthe art. These families include amino acids with basic side chains(e.g., lysine, arginine, histidine), acidic side chains (e.g., asparticacid, glutamic acid), uncharged polar side chains (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutapolynucleotidesis. The resultant mutants can be screenedfor biological activity to identify mutants that retain activity.

The term “biomarker” as used herein is a molecule, the level of whosenucleic acid or protein product has a quantitatively differentialconcentration or level with respect to an aspect of a biological statein a subject. The level of the biomarker can be measured on both thenucleic acid level and the polypeptide level. At the nucleic acid level,a nucleic acid gene or a transcript which is transcribed from any partof the subject's chromosomal and extrachromosomal genome including forexample the mitochondrial genome may be measured. Preferably an RNAtranscript, more preferably an RNA transcript includes a primarytranscript, a spliced transcript, an alternatively spliced transcript,or an mRNA of the biomarker is measured. At the polypeptide level, aprepropeptide, a propeptide, a mature peptide or a secreted peptide ofthe biomarker may be measured. A biomarker can be used either solely orin conjunction with one or more other identified biomarkers, so as toallow correlation to the biological state of interest as defined herein.

The term “biological state” as used herein to mean the result of theoccurrence of a series of biological processes. As the biologicalprocesses change relative to each other, the biological state alsoundergoes changes. One measurement of a biological state, is the levelof activity of biologic variables such as biomarkers, parameters, and/orprocesses at a specified time and under specified experimental orenvironmental conditions. A biological state can include, for example,the state of an individual cell, an organ, a tissue, and/or amulti-cellular organism. A biological state can be measured in samplestaken from a normal subject or a disease subject. Therefore, measuringthe biological state at different time intervals may indicate the stateof disease progression in a subject. The term biological state thusincludes a state that is indicative of a disease (e.g., diagnosis ordiagnosing), a state that is indicative of the progression or regressionof a disease (prognosis), a state that is indicative of thesusceptibility (risk) of a subject to a disease; and a state that isindicative of the efficacy of a treatment for a disease.

The term biological state is also used herein to refer to clinical signsand diagnostic criteria associated with a disease state. The biologicalstate of a disease state can be quantified with measurements ofbiological variables. For example, for the disease state of diabetes,the biological variables can include fasting plasma glucose, casualplasma glucose, or oral glucose tolerance test (OGTT) value.

The term “reference pattern of the disease state” is used herein to meana set of biological variables that are measured in a diseased biologicalsystem under specified experimental conditions. For example, themeasurements may be performed on blood samples at a specified timefollowing a particular glucose or insulin stimulus.

The term “baseline level or a “control level” of a biomarker expressionor activity refers to the level against which biomarker expression inthe test sample can be compared.

The term “cancer” in an individual refers to the presence of cellspossessing characteristics typical of cancer-causing cells, such asuncontrolled proliferation, immortality, metastatic potential, rapidgrowth and proliferation rate, and certain characteristic morphologicalfeatures. Often, cancer cells will be in the form of a tumor, but suchcells may exist alone within an individual, or may circulate in theblood stream as independent cells, such as leukemic cells.

The term “prostate cancer” means a cancer of the prostate tissue.

The phrase “USP2a disease or condition” refers to a disease or disorderin which USP2a is implicated. Examples of USP2a diseases or disordersinclude, but are not limited to cancers, metabolic syndromes, and thelike. Prostate and/or breast cancer are examples of USP2a diseases orconditions.

As used herein, the term “efficacy” refers to either inhibition to someextent, of cell growth causing or contributing to a cell proliferativedisorder, or the inhibition, to some extent, of the production offactors (e.g., growth factors) causing or contributing to a cellproliferative disorder. A “therapeutic efficacy” refers to relief of oneor more of the symptoms of a cell proliferative disorder. In referenceto the treatment of a cancer, a therapeutic efficacy refers to one ormore of the following: 1) reduction in the number of cancer cells; 2)reduction in tumor size; 3) inhibition (i.e., slowing to some extent,preferably stopping) of cancer cell infiltration into peripheral organs;4) inhibition, to some extent, of tumor growth; and/or 5) relieving tosome extent one or more of the symptoms associated with the disorder. Inreference to the treatment of a cell proliferative disorder other than acancer, a therapeutic efficacy refers to 1) either inhibition to someextent, of the growth of cells causing the disorder; 2) the inhibition,to some extent, of the production of factors (e.g., growth factors)causing the disorder; and/or 3) relieving to some extent one or more ofthe symptoms associated with the disorder.

As used herein, the term “sample” or “biological sample” refers to awhole organism or a subset of its tissues, cells or component parts(e.g. body fluids, including but not limited to blood, antiserum, mucus,lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amnioticfluid, amniotic cord blood, urine, vaginal fluid and semen). A sample ora biological sample further refers to a homogenate, lysate or extractprepared from a whole organism or a subset of its tissues, cells orcomponent parts, or a fraction or portion thereof, including but notlimited to, for example, plasma, serum, spinal fluid, lymph fluid, theexternal sections of the skin, respiratory, intestinal, andgenitourinary tracts, tears, saliva, milk, blood cells, tumors, organs.Most often, the sample has been removed from an animal, but the termsample or biological sample can also refer to cells or tissue analyzedin vivo, i.e., without removal from animal. Typically, sample orbiological sample will contain cells from the animal, but the term canalso refer to non-cellular biological material, such as non-cellularfractions of blood, saliva, or urine, that can be used to measure thecancer-associated polynucleotide or polypeptides levels. A sample or abiological sample further refers to a medium, such as a nutrient brothor gel in which an organism has been propagated, which contains cellularcomponents, such as proteins or nucleic acid molecules.

The term “autologous” means that the sample is obtained from the samesubject from which the sample to be evaluated is obtained.

The term “subject” refers to a vertebrate, which is preferably a mammal,more preferably a primate and still more preferably a human. Mammalsinclude, but are not limited to, primates, humans, farm animals, sportanimals, and pets.

The term “patient” refers to a subject who requires or is in need oftreatment, is receiving treatment, will receive treatment, and/or onewho is under care by a medically trained professional for a particulardisease or condition.

The term “condition” refers to the status of any cell, organ, organsystem or organism. Conditions may reflect a disease state or simply thephysiologic presentation or situation of an entity. Conditions may becharacterized as phenotypic conditions such as the macroscopicpresentation of a disease or genotypic conditions such as the underlyinggene or protein expression profiles associated with the condition.Conditions may be benign or malignant.

The term “treating” as used herein, unless otherwise indicated, meansreversing, alleviating, inhibiting the progress of, or preventing,either partially or completely, the growth of a disease or condition.The term “treatment” as used herein, unless otherwise indicated, refersto the act of treating.

The term “predicting” means a statement or claim that a particular eventwill, or is very likely to, occur in the future.

The term “prognosing” means a statement or claim that a particularbiologic event will, or is very likely to, occur in the future.Prognosis may also signal a particular outcome of a biological event,e.g., survival times.

The term “progression” means the advancement or worsening of or toward adisease or condition.

The term “cell growth” is principally associated with growth in cellnumbers, which occurs by means of cell reproduction (i.e. proliferation)when the rate of the latter is greater than the rate of cell death (e.g.by apoptosis or necrosis), to produce an increase in the size of apopulation of cells, although a small component of that growth may incertain circumstances be due also to an increase in cell size orcytoplasmic volume of individual cells. An agent that inhibits cellgrowth can thus do so by either inhibiting proliferation or stimulatingcell death, or both, such that the equilibrium between these twoopposing processes is altered.

The term “tumor growth” as used herein, unless otherwise indicated, isused as commonly used in oncology, where the term is principallyassociated with an increased mass or volume of the tumor primarily as aresult of tumor cell growth.

The term “carrier” as used herein refers to a diluent, adjuvant,excipient, or vehicle with which an active ingredient, e.g., a nucleicacid, peptide or antibody of the invention is administered. Suchcarriers can be sterile liquids, such as water and oils.

The phrase a “pharmaceutically acceptable carrier” refers to a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredient(s). A pharmaceuticallyacceptable carrier also refers to molecular entities and compositionsthat are physiologically tolerable and do not typically produce untowardreactions when administered to a human. Preferably, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The characteristics of the carrier will dependon the route of administration. Suitable pharmaceutical carriers aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin, 20thEdition.

The term “immunoassay” refers to a test that uses the binding ofantibodies to antigens to identify and measure certain substances.Immunoassays often are used to diagnose disease, and test results canprovide information about a disease that may help in planning treatment.An immunoassay takes advantage of the specific binding of an antibody toits antigen. Monoclonal antibodies are often used as they usually bindonly to one site of a particular molecule, and therefore provide a morespecific and accurate test, which is less easily confused by thepresence of other molecules. The antibodies used must have a highaffinity for the antigen of interest, because a very high proportion ofthe antigen must bind to the antibody in order to ensure that the assayhas adequate sensitivity.

The term “PCR” or “RT-PCR”, abbreviates for polymerase chain reactiontechnologies, as used here refers to technique for the detection ordetermination of nucleic acid levels, whether synthetic or expressed.

The terms “immunohistochemical” or abbreviated “IHC” as used hereinrefer to the process of detecting antigens (e.g., proteins) in abiological sample by exploiting the binding properties of antibodies toantigens in said biological sample.

The term “enzyme linked immunosorbent assay” or abbreviated “ELISA” asused herein refers to a technique for the detecting antigens (e.g.,proteins) in a biological sample.

The term “radioimmunoassay” or abbreviated “RIA” as used herein refersto a technique for the detecting antigens (e.g. proteins) in abiological sample.

USP2a

The present invention provides USP2a peptides and antibodiesspecifically immunoreactive to these USP2a peptides. Also provided arenucleic acids encoding the peptides and antibodies of the invention, aswell as probes and primers which hybridize to USP2a peptides orproteins. The present invention also provides for methods for using theUSP2a peptides and antibodies. The peptides, antibodies, and nucleicacids may be used in researching USP2a-related biological activities,e.g., signal transduction, as well as in disease diagnosis, monitoring,prognosis and therapy.

Isolated USP2a Peptides

The present invention provides isolated USP2a peptides and theirvariants and/or derivatives, as well as compositions containing two ormore USP2a peptides, variants or derivatives.

Preferably, the USP2a peptide of the present invention is or contains anepitope for the production of an antibody specifically immunoreactive tothe USP2a peptide.

In a preferred embodiment, the peptide of the present inventioncomprises a peptide containing or comprising an epitope selected from animmunogenic epitope or an antigenic epitope.

In one embodiment, the USP2a peptide of the invention is synthesized bymethods known in the art and as described below. In another preferredembodiment, the USP2a peptide is produced by expressing a nucleic acidencoding the peptide.

USP2a peptides can be synthesized by different methods well known in theart. For example, ribosomally-directed fermentation methods, as well asnon-ribosomal strategies and chemical synthesis methods. USP2a peptidescontaining the 20 natural amino acids can be prepared via recombinantexpression systems that utilize the ribosomally directed peptidesynthesis machinery of a host organism, e.g., E. coli. Alternatively,USP2a peptides, including those containing unnatural ornon-proteninogenic amino acids or modified amino acid side chains can beprepared through a solution-phase chemical synthesis of peptides (e.g.,using N-Boc protection and the activated ester route). Protocols forsequence solution-phase chemical synthesis of peptides have beendescribed in Andersson et al., Biopolymers 55:227-250 (2000). One methodused for generating peptides is a solution-phase chemical synthesis,which employs a N-tert-butoxy (N-Boc) protected amino acid and aC-protected amino acid (Andersson et al., Biopolymers 55: 227-250(2000)). An alternative solution-phase method for chemically-catalyzedpeptide synthesis employs pre-activated esters as the carboxyl componentfor coupling (Andersson et al., Biopolymers 55: 227-250 (2000)). Inaddition, enzyme-mediated solid-phase peptide synthesis has also beenemployed. Solid-phase peptide synthesis (SPPS) uses insoluble resinsupports, and has simplified and accelerated peptide synthesis andfacilitated purification (Merrifield, R. B., J. Am. Chem. Soc. 85:2149-2154 (1963)). Since the growing peptide is anchored on an insolubleresin, unreacted soluble reagents can be removed by simple filtration orwashing without manipulative losses. Solid phase peptide synthesis canbe performed using automation. Those skilled in the art will recognizethat various peptides are within the spirit and scope of the presentinvention.

In another embodiment, the USP2a peptides of the present invention canbe modified, for example, by the addition of an acetyl or amine group oramino acids at the amino- and/or carboxy-terminus of the peptide. Aminoacid addition modifications may also be performed, for example, to alterthe conformation of the epitope bearing peptide such that the epitopewill have a conformation more closely related to the structure of theepitope in the native protein. An example of a modified epitope-bearingpeptide of the invention is a peptide in which one or more cysteineresidues have been added to the peptide to allow for the formation of adisulfide bond between two cysteines, thus resulting in a stable loopstructure of the epitope-bearing peptide under non-reducing conditions.Disulfide bonds can form between a cysteine residue added to the peptideand a cysteine residue of the naturally-occurring epitope, or betweentwo cysteines which have both been added to the naturally-occurringepitope-bearing peptide.

In addition, it is possible to modify one or more amino acid residues ofthe peptide by substitution with cysteines to promote the formation ofdisulfide bonded loop structures. Cyclic thioether molecules ofsynthetic peptides can be routinely generated using techniques known inthe art, e.g., as described in PCT publication WO 97/46251, incorporatedin its entirety by reference herein. Other modifications ofepitope-bearing peptides contemplated by this invention includebiotinylation.

In one embodiment, the USP2a peptide of the present invention ismodified by adding an acetyl group at the amino terminus and/or an amidegroup at the carboxyl terminus.

The USP2a peptide of the invention may be provided as a chimericpeptide, such as in the form of a fusion peptide. For instance, theUSP2a peptide can be provided as a recombinant fusion peptide whichincludes a second peptide portion having an amino acid sequenceunrelated (heterologous) to the USP2a peptide. For example, the secondpeptide portion may be glutathione-S-transferase, or a peptide with anenzymatic activity such as alkaline phosphatase, or an epitope tag.

In one embodiment, the USP2a peptide of the present invention has abiological activity, e.g., the ability to bind to a ligand. For example,the USP2a peptide may compete with a USP2a protein in binding to theligand, thus specifically modulates the activities of the USP2a protein.

In one embodiment, the USP2a peptide contains an amino acid sequencethat is identical with or homologous to a sequence represented by eitherSEQ ID NO. 1 or SEQ ID NO. 2. A homologous sequence is at least 70%,75%, 80%, 85%, 90%, or 95% identical to the peptide represented byeither SEQ ID NO. 1 or SEQ ID NO. 2.

TABLE 1 USP2a Peptides Peptide 1 SEQ ID NO. 1 LTRPRTYGPSSLLDYDRGRPLPeptide 2 SEQ ID NO. 2 GGGK RAESQTRGTE RPLGS

In a preferred embodiment, the USP2a peptide is encoded by a nucleicacid containing any combination of nucleotide degeneracy. The USP2apeptides of SEQ ID NO. 1 and SEQ ID NO. 2 are derived from highlyhydrophilic regions (identified by Kyle-Doolittle plots) of theN-terminal portion of USP2a, and are not present in the isoform USP2b.

The present invention also provides a mixture of two or more USP2apeptides, each containing an amino acid sequence that is identical withor homologous to a sequence comprising SEQ ID NO. 1 or SEQ ID NO. 2. Inone embodiment, the mixture contains two or more peptides, eachcontaining an amino acid sequence that is identical with or homologousto a sequence comprising SEQ ID NO. 1 or SEQ ID NO. 2.

The peptides may be derivatized e.g., by conjugation with bovine serumalbumin (BSA), or keyhole limpet hemocyanin (KLH), and/or with afunctional group such as hydroxy (—OH), acetyl (—CH₂C00H) or amide(—NH₂).

Antibodies Against USP2a Peptides

In one embodiment, the present invention provides USP2a antibodies thatare specifically immunoreactive to peptides, e.g., the USP2a peptidesand USP2a proteins, their variants or derivatives as described above.The antibodies may be polyclonal or monoclonal or recombinant, producedby methods known in the art or as described in this application.

In a further embodiment, the antibodies of the present invention may belabeled with a detectable label known by one of skill in the art. Thelabel can be a radioisotope, fluorescent compound, chemiluminescentcompound, enzyme, or enzyme co-factor, or any other labels known in theart. In some aspects, the antibody that binds to an entity one wishes tomeasure (the primary antibody) is not labeled, but is instead detectedby binding of a labeled secondary antibody that specifically binds tothe primary antibody. Antibodies of the present invention include, butare not limited to, polyclonal, monoclonal, multispecific, human,humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′) fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Idantibodies to antibodies of the invention), intracellularly madeantibodies (i.e., intrabodies), and epitope-binding fragments of any ofthe above. The antibodies of the present invention can be from anyanimal origin including birds and mammals. Preferably, the antibodiesare of human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat,guinea pig, camel, horse, or chicken origin. The antibodies of thepresent invention can be monospecific or multispecific (e.g.,bispecific, trispecific, or of greater multispecificity). Multispecificantibodies can be specific for different epitopes of a peptide of thepresent invention, or can be specific for both a peptide of the presentinvention, and a heterologous epitope, such as a heterologous peptide orsolid support material. (See, e.g., WO 93/17715; WO 92/08802; WO91/00360; WO 92/05793; Tutt et al., 1991, J. Immunol., 147:60-69; U.S.Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; andKostelny et al., 1992, J. Immunol., 148:1547-1553). For example, theantibodies may be produced against a peptide containing repeated unitsof a USP2a peptide sequence of the present invention, or they may beproduced against a peptide containing two or more USP2a peptidesequences of the present invention, or the combination thereof.

In another embodiment, antibodies can be prepared from any region of thepeptides and USP2a peptides of the present invention. In addition, if apolypeptide is a receptor protein, e.g., a receptor USP2a, antibodiescan be developed against an entire receptor or portions of the receptor,for example, an intracellular domain, an extracellular domain, theentire transmembrane domain, specific transmembrane segments, any of theintracellular or extracellular loops, or any portions of these regions.Antibodies can also be developed against specific functional sites, suchas the site of ligand binding, or sites that are glycosylated,phosphorylated, myristylated, or amidated, for example.

In the present invention, the USP2a peptides for generating antibodiespreferably contain a sequence of at least 4, at least 5, at least 6, atleast 7, more preferably at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, and, preferably,between about 5 to about 50 amino acids in length, more preferablybetween about 10 to about 30 amino acids in length, even more preferablybetween about 10 to about 20 amino acids in length. The preferred USP2apeptides are those derived from the RTK proteins listed in Table 1above; that is, preferred USP2a peptides have an amino acid sequence thesame as or homologous to a portion of the sequence of the proteinslisted in Table 1.

The monoclonal antibodies of the present invention can be prepared usingwell-established methods known by those skilled in the art. In oneembodiment, the monoclonal antibodies are prepared using hybridomatechnology, such as those described by Kohler and Milstein (1975),Nature, 256:495. In a hybridoma method, a mouse, hamster, or otherappropriate host animal, is typically immunized with an immunizing agent(e.g., a USP2a peptide of the invention) to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro. The lymphocytes are then fused with an immortalizedcell line using a suitable fusing agent, such as polyethylene glycol, toform a hybridoma cell (Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, (1986) pp. 59-1031. Immortalized cell linesare usually transformed mammalian cells, particularly myeloma cells ofrodent, rabbit, bovine and human origin. Usually, rat or mouse myelomacell lines are employed. The hybridoma cells may be cultured in asuitable culture medium that preferably contains one or more substancesthat inhibit the growth or survival of the unfused, immortalized cells.For example, if the parental cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (“HAT medium”), which substances prevent the growth ofHGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies (Kozbor, J. Immunol. (1984), 133:3001; Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies. Preferably, thebinding specificity (i.e., specific immunoreactivity) of monoclonalantibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known by those skilled in the art. Thebinding specificity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson and Pollard (1980), Anal.Biochem., 107:220.

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

In another embodiment, the monoclonal antibodies of the presentinvention can also be made by recombinant DNA methods, such as thosedescribed in U.S. Pat. No. 4,816,567, which is hereby incorporated byreference in its entirety. DNA encoding the monoclonal antibodies of theinvention can be readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains ofmurine antibodies). The hybridoma cells of the invention serve as apreferred source of DNA. Once isolated, the DNA can be placed intoexpression vectors, which are then transfected into host cells such assimian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells. TheDNA also can be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567) or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the invention, or can be substituted for thevariable domains of one antigen-combining site of an antibody of theinvention to create a chimeric bivalent antibody.

In another embodiment, polyclonal antibodies of the present inventioncan also be produced by various procedures known by those skilled in theart. For the production of polyclonal antibodies in vivo, host animals,such as rabbits, rats, mice, sheep, or goats, are immunized with eitherfree or carrier-coupled peptides, for example, by intraperitoneal and/orintradermal injection. Injection material is typically an emulsioncontaining about 100 μg of peptide or carrier protein. Various adjuvantscan also be used to increase the immunological response, depending onthe host species. Adjuvants include, but are not limited to, Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants arealso well known in the art. Several booster injections may be needed,for instance, at intervals of about two weeks, to provide a useful titerof antibody which can be detected, for example, by ELISA assay usingfree peptide adsorbed to a solid surface. The titer of antibodies inserum from an immunized animal can be increased by selection ofantibodies, e.g., by adsorption of the peptide onto a solid support andelution of the selected antibodies according to methods well known inthe art.

Antibodies encompassed by the present invention can also be generatedusing various phage display methods known by those skilled in the art.In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds to the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured onto a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv, or disulfidestabilized antibody domains recombinantly fused to either the phagepolynucleotide III or polynucleotide VIII protein. Examples of phagedisplay methods that can be used to make the antibodies of the presentinvention include those disclosed in Brinkman et al. (1995) J. Immunol.Methods, 182:41-50; Ames et al. (1995) J. Immunol. Methods, 184:177-186;Kettleborough et al. (1994) Eur. J. Immunol., 24:952-958; Persic et al.(1997) Gene, 187:9-18; Burton et al. (1994) Advances in Immunology,57:191-280; PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108,each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below.

Examples of techniques that can be used to produce antibody fragmentssuch as single-chain Fvs and antibodies include those described in U.S.Pat. Nos. 4,946,778 and 5,258, 498; Huston et al. (1991) Methods inEnzymology, 203:46-88; Shu et al. (1993) Proc. Natl. Acad. Sci. USA,90:7995-7999; and Skerra et al. (1988) Science, 240:1038-1040, each ofwhich is incorporated herein by reference in its entirety.

For some uses, including the in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanized,or human antibodies. A chimeric antibody is a molecule in whichdifferent portions of the antibody are derived from different animalspecies, such as antibodies having a variable region derived from amurine monoclonal immunoglobulin and a human immunoglobulin constantregion. Methods for producing chimeric antibodies are known in the art.(See, e.g., Morrison (1985), Science, 229:1202; Oi et al. (1986),BioTechniques, 4:214; Gillies et al. (1989), J. Immunol. Methods,125:191-202; and U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397,which are incorporated herein by reference in their entirety).

Humanized antibodies are antibody molecules from non-human species thatbind to the desired antigen and have one or more complementaritydetermining regions (CDRs) from the nonhuman species and frameworkregions from a human immunoglobulin molecule. Often, framework residuesin the human framework regions are substituted with correspondingresidues from the CDR and framework regions of the donor antibody toalter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding, and bysequence comparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. Nos. 5,693,762 and5,585,089; and Riechmann et al. (1988) Nature, 332:323, which areincorporated herein by reference in their entireties). Antibodies can behumanized using a variety of techniques known in the art, including, forexample, CDR-grafting (EP 239, 400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089); veneering or resurfacing(EP 592,106; EP 519,596; Padlan (1991), Molecular Immunology,28(4/5):489-498; Studnicka et al. (1994) Protein Engineering,7(6):805-814; Roguska et al. (1994) Proc. Natl. Acad. Sci. USA,91:969-973; and chain shuffling (U.S. Pat. No. 5,565,332); each of whichare incorporated herein by reference in their entirety.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients, so as to avoid or alleviate immune reactionto foreign protein. Human antibodies can be made by a variety of methodsknown in the art, including the phage display methods described above,using antibody libraries derived from human immunoglobulin sequences.See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publicationsWO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741; each of which is incorporated herein byreference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin polynucleotides. For example, the humanheavy and light chain immunoglobulin polynucleotide complexes can beintroduced randomly, or by homologous recombination, into mouseembryonic stem cells. Alternatively, the human variable region, constantregion, and diversity region may be introduced into mouse embryonic stemcells, in addition to the human heavy and light chain polynucleotides.The mouse heavy and light chain immunoglobulin polynucleotides can berendered nonfunctional separately or simultaneously with theintroduction of human immunoglobulin loci by homologous recombination.In particular, homozygous deletion of the J_(H) region preventsendogenous antibody production. The modified embryonic stem cells areexpanded and microinjected into blastocysts to produce chimeric mice.The chimeric mice are then bred to produce homozygous offspring whichexpress human antibodies. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of apolypeptide of the invention.

Thus, using such a technique, it is possible to produce useful humanIgG, IgA, IgM, IgD and IgE antibodies. For an overview of the technologyfor producing human antibodies, see Lonberg and Huszar (1995) Intl. Rev.Immunol., 13:65-93. For a detailed discussion of the technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., PCT publications WO 98/24893;WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877;U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;5,545,806; 5,814,318; 5,885,793; 5,916,771; 5,939,598; 6,075,181; and6,114,598, each are incorporated by reference herein in their entirety.In addition, companies such as Abgenix, Inc. (Fremont, Calif.), ProteinDesign Labs, Inc. (Mountain View, Calif.) and Genpharm (San Jose,Calif.) can be engaged to provide human antibodies directed against aselected antigen using technology similar to the above describedtechnologies.

Once an antibody molecule of the present invention has been produced byan animal, a cell line, chemically synthesized, or recombinantlyexpressed, it can be purified (i.e., isolated) by any method known inthe art for the purification of an immunoglobulin or polypeptidemolecule, for example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen, Protein A, and sizingcolumn chromatography), centrifugation, differential solubility, or byany other standard technique for the purification of proteins. Inaddition, the antibodies of the present invention or fragments thereofcan be fused to heterologous polypeptide sequences described herein orotherwise known in the art, to facilitate purification.

In one embodiment, the present invention provides antibodies thatspecifically immuoreact to a tyrosine USP2a protein, or fragment orvariant thereof.

In one embodiment, the present invention provides a novel monoclonalantibody that specifically recognizes a sequence selected from the groupconsisting of SEQ ID NO. 1 SEQ ID NO. 2 and variants thereof.

The present invention further provides a mixture containing two or moremonoclonal antibodies produced as described above. In a preferredembodiment, the mixture contains two or more monoclonal antibodiesraised against different USP2a peptides derived from the same USP2aprotein. In another preferred embodiment, the mixture contains two ormore monoclonal antibodies raised against different USP2a peptides, atleast two of which are derived from different USP2a proteins.

In one embodiment, the antibody mixture contains two or more antibodiesraised against peptides containing the amino acid sequence selected fromthe group consisting of SEQ ID NOs. 1 and 2.

In a preferred embodiment, specifically immunoreactive antibodiesinclude those with a dissociation constant or Kd of less than about5×10⁻², 1×10⁻², 5.times×10⁻³, 1×10⁻³, 5×10⁻⁴, or 1×10⁻⁴. In a morepreferred embodiment, specifically immunoreactive antibodies includethose with a dissociation constant or Kd less than about 5×10⁻⁵, 1×10⁻⁵,5×10⁻⁶, 1×10⁻⁶, 5×10⁻⁷, 1×10⁻⁷, 5×10⁻⁸, or 1×10⁻⁸. In an even morepreferred embodiment, specifically immunoreactive antibodies includethose with a dissociation constant or Kd of less than about 5×10⁻⁹,1×10⁻⁹, 5×10⁻¹⁰, 1×10⁻¹⁰, 5×10⁻¹¹, 10×10⁻¹¹, 5×10⁻¹², 1×10⁻¹², 5×10⁻¹³,1×10⁻¹³, 5×10⁻¹⁴, 1×10⁻¹⁴, 5×10⁻¹⁵, or 1×10⁻¹⁵.

Nucleic Acids

In one embodiment, the present invention provides a nucleic acidencoding a peptide of the present invention, or a complementary sequencethereof.

In another embodiment, the present invention provides a mixturecontaining two or more nucleic acids as described above. In a furtherembodiment, the present invention also provides a probe and/or a primercomprising an isolated oligonucleotide, wherein the oligonucleotidecomprises a region of nucleotide sequence which hybridizes understringent conditions to at least 12 consecutive nucleotides of a nucleicacid coding a USP2a peptide of the present invention, or theircomplementary sequences thereof, or naturally occurring mutants thereof.

In one embodiment, the present invention provides a mixture containingtwo or more probes and/or primers as described above. In one embodiment,the present invention also provides nucleic acids encoding an antibodyof the present invention (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof). Ina specific embodiment, a nucleic acid of the present invention encodesan antibody (including molecules comprising, or alternatively consistingof, antibody fragments or variants thereof), comprising, oralternatively consisting of, a V_(H) domain having an amino acidsequence of any one of the V_(H) domains of a heavy chain expressed byan anti-USP2a protein antibody-expressing cell line of the presentinvention (e.g., a hybridoma) and a V_(L) domain having an amino acidsequence of a light chain expressed by an anti-USP2a proteinantibody-expressing cell line of the present invention. In anotherembodiment, a nucleic acid molecule of the present invention encodes anantibody (including molecules comprising, or alternatively consistingof, antibody fragments or variants thereof), comprising, oralternatively consisting of, a V_(H) domain having an amino acidsequence of any one of the V_(H) domains of a heavy chain expressed byan anti-USP2a protein antibody-expressing cell line of the presentinvention, or a V_(L) domain having an amino acid sequence of a lightchain expressed by an anti-USP2a protein antibody-expressing cell lineof the present invention.

In a further embodiment, two or more nucleic acids encoding an antibodyof the present invention may be provided in a mixture. Standardtechniques known to those of skill in the art can be used to introducemutations in the nucleotide sequence encoding a molecule of the presentinvention, including, for example, site-directed mutapolynucleotidesisand PCR-mediated mutapolynucleotidesis which result in amino acidsubstitutions. This can be used to prepare desired antibodies, e.g.,humanized antibodies as described herein. In a preferred embodiment, thevariants (including derivatives) encode less than 50 amino acidsubstitutions, less than 40 amino acid substitutions, less than 30 aminoacid substitutions, less than 25 amino acid substitutions, less than 20amino acid substitutions, less than 15 amino acid substitutions, lessthan 10 amino acid substitutions, less than 5 amino acid substitutions,less than 4 amino acid substitutions, less than 3 amino acidsubstitutions, or less than 2 amino acid substitutions, relative to thereference V_(H) domain, V_(H) CDR1, V_(H) CDR2, V_(H) CDR3, V_(L)domain, V_(L) CDR1, V_(L) CDR2, or V_(L) CDR3. In one embodiment,nucleic acids encoding epitopes can be recombined with a second nucleicacid as an epitope tag (e.g., the hemagglutinin (“HA”) tag or flag tag)to aid in detection and purification of the expressed polypeptide. Forexample, a system for the ready purification of non-denatured fusionproteins expressed in human cell lines has been described by Janknechtet al., (1991, Proc. Natl. Acad. Sci. USA, 88:8972-897). In this system,the second nucleic acid is subcloned into a vaccinia recombinationplasmid such that the open reading frame of the polynucleotide istranslationally fused to an amino-terminal tag having six histidineresidues. The tag serves as a matrix binding domain for the fusionprotein. Extracts from cells infected with the recombinant vacciniavirus are loaded onto an Ni.sup.2+ nitriloacetic acid-agarose column andhistidine-tagged proteins are selectively eluted withimidazole-containing buffers.

If a clone containing a nucleic acid encoding a particular antibody isnot available, but the sequence of the antibody molecule is known, anucleic acid encoding the immunoglobulin can be chemically synthesizedor obtained from a suitable source (e.g., an antibody cDNA library, or acDNA library generated from, (or a nucleic acid, preferably poly A+ RNA,isolated from), any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody of the invention by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence. Alternatively, cloning using an oligonucleotide probespecific for the particular nucleic acid sequence to be identified,e.g., a cDNA clone from a cDNA library that encodes the desired antibodycan be employed. Amplified nucleic acids generated by PCR can then becloned into replicable cloning vectors using any method well known inthe art.

Applications

The USP2a peptides, nucleic acids encoding the USP2a peptides,antibodies specifically immunoreactive with the USP2a peptides, as wellas nucleic acids of the present invention are useful for research andfor disease detection, diagnosis, prognosis and treatment of certaintypes of cancer, in particular, prostate cancer.

As nonlimiting examples, antibodies of the present invention can be usedto purify, detect, and target the USP2a polypeptides of the presentinvention, including both in vitro and in vivo diagnostic, detection,screening, and/or therapeutic methods. For example, the probes, primersand antibodies can be used in immunoassays for qualitatively andquantitatively measuring levels of USP2a biomarkers in biologicalsamples. (See, e.g., Harlow et al., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 2nd Ed. 1988, which is incorporatedby reference herein in its entirety).

In one embodiment, probes, primers and antibodies of the invention canbe used as a part of a diagnostic test kit for identifying dysfunctionsassociated with mis-expression of a USP2a protein containing thesequence of the USP2a peptide of the present invention, such as fordetecting in a sample of cells isolated from a patient, a level of aUSP2a protein, a level of a nucleic acid encoding a USP2a protein; e.g.measuring a USP2a mRNA level in a cell, or determining whether a genomicUSP2a gene has been mutated or deleted. These probes and primers of thepresent invention can also be used as a part of “antisense” therapywhich refers to administration or in situ generation of oligonucleotideprobes or their derivatives which specifically hybridize (e.g. bind)under cellular conditions, with the cellular mRNA and/or genomic DNAencoding one or more of the subject USP2a proteins so as to inhibitexpression of that protein, e.g. by inhibiting transcription and/ortranslation. In a further embodiment, the antibodies of the presentinvention can also be used for therapies as described below.

In one embodiment, the USP2a peptides of the present invention may serveas biomarkers for (1) diagnosis of USP2a related diseases or conditions(e.g., cancers, hyperproliferative conditions, metabolic syndromes, etc)(2) prognosis of diseases (e.g., monitoring disease progression orregression from one biological state to another), (3) determination ofsusceptibility (risk) of a subject to diseases, or (4) evaluation of theefficacy of a treatment for a disease. Specific biomarkers of thepresent invention include, but are not limited to, target USP2apeptides, their variants, derivatives or resultant antibodies asdescribed above. In one embodiment, the biological state can bemathematically defined by the values of x and p at a given time, asknown in the art. Once a biological state of the model is mathematicallyspecified, numerical integration of the above equation using a computerdetermines, for example, the time evolution of the biological variablesx(t) and hence the evolution of the biological state over time. In afurther embodiment, measurements may be performed on biopsy samples, orcell cultures derived from a diseased human or animal. Examples ofdiseased biological systems include cellular or animal models of thedisease or a patient.

In one embodiment, the baseline level can be a “normal level” (i.e.,level in a sample from a normal subject). Therefore, it can bedetermined, based on the control or baseline level of biomarkerexpression or biological activity, whether a sample to be evaluated fordisease cell growth has a measurable increase, decrease, orsubstantially no change in biomarker expression as compared to thebaseline level. In another embodiment, the baseline level can be a“negative control” which herein refers to a baseline level establishedin a sample from the subject or from a population of individuals whichis believed to be normal (i.e., non-tumorous, not undergoing neoplastictransformation, not exhibiting inappropriate cell growth). In anotherembodiment, a baseline can be indicative of a positive diagnosis ofdisease. Such a baseline level, also referred to herein as a “positivecontrol” baseline, refers to a level of biomarker expression orbiological activity established in a sample from the subject, anothersubject, or a population of individuals, wherein the sample wasbelieved, based on data for that cell sample, to have the disease (i.e.,tumorous, exhibiting inappropriate cell growth, cancerous). In anotherembodiment, the baseline level can be established from a previous sampleof the subject being tested, so that the disease progression orregression of a subject can be monitored over time and/or so that theefficacy of a given therapeutic protocol can be evaluated over time. Ina preferred embodiment, the method for establishing a baseline level ofthe biomarker expression is selected based on the sample type, thetissue or organ from which the sample is obtained, the status of thesubject to be evaluated, and, as discussed above, the focus or goal ofthe assay (e.g., diagnosis, staging, monitoring). In a more preferredembodiment, the method is the same method that will be used to evaluatethe sample in the subject. In a most preferred embodiment, the baselinelevel is established using the same cell type as the cell to beevaluated.

In one embodiment, the baseline level of biomarker expression orbiological activity is established in an autologous control sampleobtained from the subject. The autologous control sample can be a sampleof isolated cells, a tissue sample or a bodily fluid sample, and ispreferably a cell sample or tissue sample. In a further embodiment, thecontrol sample should be of or from the same cell type. In a preferredembodiment, the control sample is obtained from the same organ, tissueor bodily fluid as the sample to be evaluated, such that the controlsample serves as the best possible baseline for the sample to beevaluated. In one embodiment, when the goal of the assay is diagnosis ofabnormal cell growth, it is desirable to take the control sample from apopulation of cells, a tissue or a bodily fluid which is believed torepresent a normal cell, tissue, or bodily fluid, or at a minimum, acell or tissue which is least likely to be undergoing or potentially bepredisposed to develop tumor cell growth. For example, if the sample tobe evaluated is an area of apparently abnormal cell growth, such as atumorous mass, the control sample is preferably obtained from a sectionof apparently normal tissue (i.e., an area other than and preferably areasonable distance from the tumorous mass) in the tissue or organ wherethe tumorous mass is growing. In one aspect, if a tumor to be evaluatedis in the colon, the test sample would be obtained from the suspectedtumor mass in the colon and the control sample would be obtained from adifferent section of the colon, which is separate from the area wherethe mass is located and which does not show signs of uncontrolledcellular proliferation.

In one embodiment, a difference in expression level between a testsample and a baseline, either measured at the nucleic acid or thepeptide level, is considered positive for the particular purpose of thetest if the difference is at least 20%, 30%, 40%, 50%, preferably atleast 70%, more preferably 80%, 90%, or even more preferably 2-fold,3-fold, 4-fold, 5-fold, 10-fold or more. In one embodiment, target USP2abiomarkers may originate from different parts of the cell and may becell surface receptors, although they can also be intracellularproteins. The USP2a proteins may be over expressed or under expressed ina sample. The level of the target gene or protein can be determined byconventional methods such as expression assays to determine the level ofexpression of the gene, by biochemical assays to determine the level ofthe gene product, or by immunoassays using antibodies reactive to USP2a.Examples of detection methods include those known to those of skill inthe art or taught in numerous texts and laboratory manuals (see forexample, Ausubel et al. (1995) Short Protocols in Molecular Biology, 3rdEd. John Wiley & Sons, Inc.). If appropriate, the target USP2a can beidentified as a cell surface molecule in tissue or in a bodily fluid,such as serum.

For example, methods of detecting nucleic acid of a USP2a biomarkerinclude but are not limited to, RNA fingerprinting, Northern blotting,polymerase chain reaction (PCR), ligase chain reaction, Q betareplicase, isothermal amplification method, strand displacementamplification, transcription based amplification systems, nucleaseprotection (SI nuclease or RNAse protection assays) as well as methodsdisclosed in WO88/10315, WO89/06700, PCT/US87/00880, PCT/US89/01025; allof which are incorporated herein by reference.

In one embodiment, detecting the expression level of a nucleic acid of aUSP2a biomarker may include (i) providing a probe or a primer of thepresent invention; (ii) contacting the probe or the primer with asample; and (iii) detecting, by hybridization of the probe or the primerto nucleic acids in the sample, the presence and absence or the level ofa USP2a biomarker nucleic acid. The method may also include amplifyingthe nucleic acid of the USP2a biomarker before detecting. For instance,the probe or the primer can be employed in a polymerase chain reaction(PCR) or in a ligation chain reaction (LCR). A difference in the levelof expression between a test sample (e.g., from a patient subject) and acontrol sample (e.g., from a normal subject) is indicative of thebiological state of interest. The probe or the primer used may also be amixture of two or more probes or primers as described above.

In a further embodiment, the nucleic acid detection method also includesdetecting, in a sample of the subject, the presence or absence of agenetic lesion spanning a region represented by a nucleic acid encodinga USP2a peptide of the present invention. Detecting the genetic lesionincludes ascertaining the existence of at least one of (i) deletion ofone or more nucleotides from a USP2a gene; an addition of one or morenucleotides to the gene, a substitution of one or more nucleotides ofthe gene, a gross chromosomal rearrangement of the gene; an alterationin the level of a messenger RNA transcript of the gene; the presence ofa non-wild type splicing pattern of a messenger RNA transcript of thegene; a non-wild type level of the protein; and/or an aberrant level ofa USP2a protein.

In alternate embodiments, the level of a USP2a protein is detected in animmunoassay using an antibody of the present invention, which isspecifically immunoreactive with the USP2a protein. Preferably, themethod of detection comprises contacting the sample with an antibody ofthe present invention (including an antibody mixture) and determiningthe presence and absence or the level of a USP2a biomarker protein. Adifference in the level of expression between a test sample (e.g., froma patient subject) and a control sample (e.g., from a normal subject) isindicative of the biological state of interest. The antibody used mayalso be a mixture of two or more antibodies, or antibodies raisedagainst a mixture of USP2a peptides as described above.

Various methods known in the art for detecting specific antibody-antigenbinding can be used. Exemplary immunoassays which can be conductedaccording to the invention include fluorescence polarization immunoassay(FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA),nephelometric inhibition immunoassay (NIA), enzyme linked immunosorbentassay (ELISA), and radioimmunoassay (RIA). Zola, Monoclonal Antibodies:A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987). In oneembodiment, a detectable label can be attached to the subject antibodiesand is selected so as to meet the needs of various uses of the methodwhich are often dictated by the availability of assay equipment andcompatible immunoassay procedures. Appropriate labels include, withoutlimitation, radionuclides (e.g., .sup.125I, .sup.131I, .sup.35S,.sup.3H, or .sup.32P), enzymes (e.g., alkaline phosphatase, horseradishperoxidase, luciferase, or .beta.-glactosidase), fluorescent moieties orproteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), orluminescent moieties (e.g., Qdot™ nanoparticles supplied by the QuantumDot Corporation, Palo Alto, Calif.). General techniques to be used inperforming the various immunoassays noted above are known to those ofordinary skill in the art.

In another embodiment of the invention, an antibody of the presentinvention (i.e., the primary antibody) needs not be labeled, and thepresence thereof can be detected using a labeled second antibody whichbinds to the primary antibody.

In one embodiment, antibodies can be arrayed on a substrate anddetection of biomarkers may be performed by antibody array method, forexample, as described in an application by the same inventor entitled“Antibody Protein Analysis Chip,” hereby incorporated by reference inits entirety.

In another embodiment, the antibodies can be used for in vivo diagnosticassays, such as in vivo imaging. Generally, the antibody is labeled witha radionuclide (such as ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, or ³H) so that thecells or tissue of interest can be localized using immunoscintiography.In a further embodiment, the antibody may also be used as stainingreagent in pathology, following techniques well known in the art.

One embodiment of the present invention is a method for the diagnosis ofa cancer that includes determining the level of USP2a in a test sample.In this method, the level of USP2a is indicative of the presence ofcancer cells. The presence of USP2a at an increased level as compared toa normal baseline control is an indication of the presence of a cancer,a possible predisposition to such cancer or a susceptibility to ananti-cancer therapeutic treatment. The level of USP2a can be determinedby conventional methods such as expression assays to determine the levelof expression of the USP2a gene (using the probes/primers, or mixturesthereof provided by the invention), by biochemical assays to determinethe level of the gene product, or by immunoassays. In one embodiment ofthis method, the level of USP2a can be determined by identifying USP2aas a cell surface molecule in tissue or by detecting USP2a in solubleform in a bodily fluid, such as serum, that can be immobilized. TheUSP2a level can be determined by contacting a patient test sample withan antibody, or a fragment thereof, that binds specifically to USP2a anddetermining whether the anti-USP2a antibody or fragment has bound to theUSP2a protein. The USP2a level can be determined by using a firstmonoclonal antibody that binds specifically to USP2a and a secondantibody that binds to the first antibody. This method can be used todetermine the prognosis for cancer in the patient or to determine thesusceptibility of the patient to a therapeutic treatment.

A further embodiment of the present invention is a method for thediagnosis of a tumor or the monitoring of tumor growth or regression ortumor therapy in a patient. The methods include determining the level ofUSP2a in a patient sample.

In one embodiment, the present invention further provides kits fordisease diagnosis, prognosis, risk assessment, and/or treatment efficacydetermination. Such kits are useful in a clinical setting for use indiagnosing a patient for a disease, monitoring the disease progression,testing patient's samples (e.g., biopsied), for example, to determine orpredict if the patient's disease (e.g., cancer) will be resistant orsensitive to a given treatment or therapy with a drug, compound,chemotherapy agent, or biological treatment agent. In a furtherembodiment, the kit also provides a predictor set comprising a nucleicacid or a nucleic acid mixture of the present invention or an antibodyor an antibody mixture of the present invention. In another embodiment,the kits may encompass desired reagents for the specific detectionmethod to be used, e.g., nucleic acid assays and immunoassays asdescribed above, and known in the art.

In a preferred embodiment, the kit preferably contains any means ofdetecting the expression or activity of USP2a in a test sample, andpreferably includes a probe, PCR primers, or a mixture of nucleic acidsof the invention, or an antibody, a mixture of antibodies of theinvention, antigen binding peptide, or fragment thereof, that binds toUSP2a. The kit can include any reagent needed to perform a diagnosticmethod envisioned herein. The antibody, or fragment thereof, can beconjugated to another unit, for example a marker or immobilized to asolid carrier (substrate). In a further embodiment, the kit can containa second antibody for the detection of USP2a:antibody complexes. In oneembodiment, the kit can contain a means for detecting a control markercharacteristic of a cell type in the test sample. The antibody, orfragment thereof, may be present in free form or immobilized to asubstrate such as a plastic dish, a test tube, a test rod and so on. Thekit can also include suitable reagents for the detection of and/or forthe labeling of positive or negative controls, wash solutions, dilutionbuffers and the like, as well as instructions.

More specifically, according to the present invention, a means fordetecting USP2a expression or biological activity can be any suitablereagent that can be used in a method for detection of USP2a expressionor biological activity as described previously herein. Such reagentsinclude, but are not limited to: a probe or primer, or a mixture ofnucleic acids of the invention, that hybridizes under stringenthybridization conditions to USP2a or a fragment thereof (including to aUSP2a-specific regulatory region in the biomarker-encoding gene); RT-PCRprimers for amplification of mRNA encoding USP2a or a fragment thereof;and/or an antibody or a mixture of antibodies of the present invention,antigen-binding fragment thereof or other antigen-binding peptide thatselectively binds to USP2a.

In another embodiment, the means for detecting a USP2a marker and/or acontrol marker of the assay kit of the present invention can beconjugated to a detectable tag or detectable label. Such tag can be anysuitable tag which allows for detection of the reagents used to detectUSP2a or control marker and includes, but is not limited to, anycomposition or label detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means, asdescribed herein and known in the art.

In a further embodiment, the means for detecting the assay kit of thepresent invention can be immobilized on a substrate. Such a substratecan include any suitable substrate for immobilization of a detectionreagent such as would be used in any of the previously described methodsof detection. Briefly, a substrate suitable for immobilization of ameans for detecting includes any solid support, such as any solidorganic, biopolymer or inorganic support that can form a bond with themeans for detecting without significantly effecting the activity and/orability of the detection means to detect the desired target molecule.Exemplary organic solid supports include, but are not limited to,polymers such as polystyrene, nylon, phenol-formaldehyde resins, acryliccopolymers (e.g., polyacrylamide), stabilized intact whole cells, andstabilized crude whole cell/membrane homogenates. Exemplary biopolymersupports include, but are not limited to, cellulose, polydextrans (e.g.,Sephadex), agarose, collagen and chitin. Exemplary inorganic supportsinclude, but are not limited to, glass beads (porous and nonporous),stainless steel, metal oxides (e.g., porous ceramics such as ZrO₂, TiO₂,Al₂O₃, and NiO) and sand.

Other Applications

In one embodiment, the present invention provides a method formodulating one or more of growth, differentiation, or survival of a cellby modulating USP2a expression or activity, e.g., by potentiating ordisrupting certain protein-protein interactions. In general, whethercarried out in vivo, in vitro, or in situ, the method comprises treatingthe cell with an effective amount of a USP2a peptide, a nucleic acid, oran antibody of the present invention, at least one of (i) rate ofgrowth, (ii) differentiation, or (iii) survival of the cell. Themodulatory effects as described herein are useful for researchers onsignal transduction pathways, as well as disease treatment.

In another embodiment, the present invention provides an antisensenucleic acid that specifically hybridizes to a nucleic acid encodingUSP2a of the present invention, wherein the antisense nucleic acidinhibits the expression of USP2a. In a further embodiment, the presentinvention provides a method of inhibiting the expression of USP2a of thepresent invention by contacting a sample in vitro, or in vivo, with anantisense nucleic acid of the present invention so that expression ofUSP2a is inhibited.

In another embodiment, an antibody of the present invention may bind toand competitively inhibit polypeptide multimerization and/or binding ofUSP2a of the present invention to a ligand, thus modulate the activityof USP2a in signal transduction.

In one embodiment, a USP2a MAb, upon binding to the corresponding USP2aprotein or peptide located on a cell membrane, induces apoptosis in thecell expressing the corresponding USP2a on its membrane. The MAb mayalso reduce the number of cells, or inhibit cell growth of the cellsthat express corresponding USP2a (known as USP2a-expressing cells). In apreferred embodiment, the reduction in cell number or inhibition of cellgrowth is by at least about 10%, about 20%, about 30%, about 40%, about50%, about 65%, about 75%, or greater. In another embodiment, theUSP2a-expressing cells are disease cells, e.g., cancer or tumor cells.In a more preferred embodiment, the cancers include but are not limitedto, colorectal cancer, pancreatic cancer, lung cancer, gastric cancer,hepatocellular carcinoma, breast cancer and thyroid cancer.

In one embodiment, the MAb may also inhibit the proliferation ofUSP2a-expressing cells, preferably by at least about 10%, about 20%,about 30%, about 40%, about 50%, about 65%, about 75%, or greater. Inanother embodiment, the MAb inhibits the cell growth of theUSP2A-expressing cells, preferably by at least about 10%, about 20%,about 30%, about 40%, about 50%, about 65%, about 75%, or greater.Preferably, the USP2a-expressing cells are disease cells, e.g., canceror tumor cells. More preferably, the cancers include but are not limitedto, colorectal cancer, pancreatic cancer, lung cancer, gastric cancer,hepatocellular carcinoma, breast cancer and thyroid cancer.

Therefore, antibodies of the present invention can act as agonists orantagonists of USP2a. For example, the present invention includesantibodies which disrupt receptor/ligand interactions with polypeptidesof the present invention either partially or fully. The presentinvention also includes receptor-specific antibodies which do notprevent ligand binding, but do prevent receptor activation. Receptoractivation (i.e., signaling) can be determined by techniques describedherein or as otherwise known in the art. For example, receptoractivation can be determined by detecting the phosphorylation (e.g., ontyrosine or serine/threonine) of the receptor or its substrate byimmunoprecipitation followed by Western Blot analysis.

In one embodiment, the method can be carried out with USP2a therapeuticssuch as a monoclonal antibody, an antisense nucleic acid, or a USP2apeptide of the present invention which agonizes or antagonizes theeffects of signaling from a USP2a protein or ligand binding of a USP2aprotein. The antisense nucleic acid of the present invention inhibitsthe expression of a target USP2a gene, while the peptide or the antibodymay competitively inhibit ligand interactions with the wild-type USP2aprotein.

In a further embodiment, the USP2a peptides of the present invention canbe introduced together with a carrier protein, such as an albumin, to ananimal system (such as rabbit or mouse). Alternatively, if the peptideis of sufficient length (e.g., at least about 15-25 amino acids), thepolypeptide can be presented without a carrier.

In one embodiment, the composition for therapy is formulated foradministration by intraperitoneal, intravenous, subcutaneous, andintramuscular injections, and other forms of administration such asoral, mucosal, via inhalation, sublingually, etc.

In one embodiment, the nucleic acids and antibodies of the presentinvention may be delivered with a carrier. Water or aqueous solution,saline solutions and aqueous dextrose and glycerol solutions arepreferably employed as carriers, particularly for injectable solutions.The carrier may also be a pharmaceutically acceptable carrier.

In another embodiment, a nucleic acid encoding any of the antibodies ofthe present invention can also be used for delivery and expression ofany of the antibodies of the present invention in a desired cell. It isapparent that an expression vector can be used to direct expression ofan antibody. The expression vector can be administered by any meansknown in the art, such as, but not limited to, intraperitoneally,intravenously, intramuscularly, subcutaneously, intrathecally,intraventricularly, orally, enterally, parenterally, intranasally,dermally, sublingually, or by inhalation. For example, administration ofexpression vectors includes local or systemic administration, includinginjection, oral administration, particle gun or catheterizedadministration, and topical administration. One skilled in the art isfamiliar with administration of expression vectors to obtain expressionof an exogenous protein in vivo. See, e.g., U.S. Pat. Nos. 6,436,908;6,413,942; and 6,376,471; each of which is incorporated by reference inits entirety.

The therapeutic polynucleotides and polypeptides of the presentinvention can be delivered using gene delivery vehicles. The genedelivery vehicle can be of viral or non-viral origin (see generally,Jolly (1994), Cancer Gene Therapy 1:51; Kimura (1994), Human GeneTherapy 5:845; Connelly (1985), Human Gene Therapy 1:185; and Kaplitt(1994), Nature Genetics 6:148). Expression of such coding sequences canbe induced using endogenous mammalian or heterologous promoters.Expression of the coding sequence can be either constitutive orregulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622; WO93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat.Nos. 5,219,740; 4,777,127; GB Patent No. 2,200,651; and EP Patent No. 0345 242; alphavirus-based vectors, e.g., Sindbis virus vectors, Semlikiforest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373;ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923;ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus(AAV) vectors, e.g., PCT Publication Nos. WO 94/12649, WO 93/03769; WO93/19191; WO 94/28938; WO 95/11984 and WO 95/00655; each of which isincorporated by reference in its entirety. Administration of DNA linkedto killed adenovirus as described in Curiel (1992), Hum. Gene Ther.3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but are not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel (1992), Hum. Gene Ther.3:147); ligand-linked DNA (see, e.g., Wu (1989), J. Biol. Chem.264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes.

Naked DNA can also be employed. Exemplary naked DNA introduction methodsare described in PCT Publication No. WO 90/11092 and U.S. Pat. No.5,580,859 herein incorporated by reference in their entirety. Liposomesthat can act as gene delivery vehicles are described in U.S. Pat. No.5,422,120; PCT Publication Nos. WO 95/13796; WO 94/23697; WO 91/14445;and EP Patent NO. 0 524 968 herein incorporated in their entirety.Additional approaches are described in Philip (1994), Mol. Cell Biol.14:2411 and in Woffendin (1994), Proc. Natl. Acad. Sci. 91:1581.

Other formulations include suitable delivery forms known in the artincluding, but not limited to, carriers such as liposomes. See, forexample, Mahato et al. (1997) Pharm. Res. 14:853-859. Liposomalpreparations include, but are not limited to, cytofectins, multilamellarvesicles and unilamellar vesicles.

In another embodiment, the present invention also contemplatesadministration of a composition comprising nucleic acids or antibodiesof the present invention conjugated to other molecules, such asdetectable labels, or therapeutic or cytotoxic agents. The agents mayinclude, but are not limited to radioisotopes, toxins, toxoids,inflammatory agents, enzymes, antisense molecules, peptides, cytokines,or chemotherapeutic agents. Methods of conjugating the antibodies withsuch molecules are generally known to those of skilled in the art. See,e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat.No. 5,314,995; and EP 396,387; the disclosures of which are incorporatedherein by reference in their entirety.

The dosage required for the treatment depends on the choice of the routeof administration, the nature of the formulation, the nature of thesubject's illness, the subject's size, weight, surface area, age andsex; other drugs being administered, and the judgment of the attendingphysician. Suitable dosages are in the range of 0.01-1000.0 mg/kg.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, microbiology, recombinant DNA, and immunology, whichare within the skill of the art.

Such techniques are explained fully in the literature and herein above.See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. bySambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press:1989); DNA Cloning, Volumes 1 and 11 (D. N. Glover ed., 1985);Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S.Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J.Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J.Higgins eds. 1984); Culture Of Animal Cells (R. 1. Freshney, Alan R.Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987,Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155(Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

The invention will be better understood by reference to the followingexample which serve to illustrate but not to limit the presentinvention.

EXAMPLES Example 1 Production and Characterization of PolyclonalAntisera for USP2a

SPF rabbits (Maine Biotechnology Services, Inc.) were used to generatepolyclonal antisera. Forty eight (48) rabbits were used. The polyclonalantibodies are referred to hereinafter as USP2a-1 and USP2a-2 and havebeen deposited with the ATCC (ATCC deposit numbers ______and ______,respectively).

Each rabbit was injected with one of the peptides of the presentinvention as shown in Table 2.

TABLE 2 Injected Peptides USP2a-1 (SEQ ID NO. 1) LTRPRTYGPSSLLDYDRGRPLUSP2a-2 (SEQ ID NO. 2) GGGK RAESQTRGTE RPLGS

The rabbits were bled, and the resulting antisera were then pooled andaffinity purified using the same epitopes against which they had beenraised. Affinity purification was carried out according to the followingprocedure:

Step 1: Affinity Column Preparation

The immunoaffinity column was prepared by coupling the peptides of SEQID NO. 1 or SEQ ID NO. 2 to 1 ml of activated sepharose beads.

Step 2: Loading of the Antisera

The antisera was loaded at a concentration of 2 μg/mL onto thepeptide-sepharose column and incubated 1 hour at 37° C.

Step 3: Elution

After several washes of the column, the elution of bound antibody wasperformed using elution buffer containing 0.02% sodium azide. Fractionscontaining the antibody were pooled and the final concentration ofimmunopurified antibody was determined by reading the optical density at280 nm using U.V. spectrophotometer.

Step 4: ELISA Test of the Immunopurified Antibody

The blocking reagent SeaBlock was loaded into the wells in a NEATconcentration and incubated for 30 minutes at 37° C. After theincubation, four samples of serum (pre-bleed Rb 1, pre-bleed Rb 2,peptide 1 and peptide 2) were added into the wells at 6 differentconcentrations. The four samples were diluted using 0.15M PBS toconcentrations of 1:50, 1:250, 1:1250, 1:6250, 1:31250, and 1:156000.Each of these concentrations of the four serums were added to the wellsthen incubated at room temperature for 30 minutes. Lastly, a secondaryantibody, anti-Rb HRP, (HRP-lot#86569) was diluted to a concentration of1:10000 using 0.15M PBS with 0.05% Tween20 and incubated at roomtemperature for 30 minutes. The final concentration of the samples, asshown in Tables 3 and 4, was determined by reading the absorbance at 450nm using the U.V. spectrophotometer.

TABLE 3 Pre-Bleed Rb 1 and 2 Sample Analysis ELISA Reactivity to ELISAReactivity to Concentration Antisera Rb1 Antisera Rb2 1:50 0.33 0.341:250 0.24 0.02 1:1250 0.36 0.03 1:6250 0.33 0.01 1:31250 0.20 0.201:156000 0.43 0.13

TABLE 4 USP2a-1 Sample Analysis ELISA Reactivity to ELISA Reactivity toAntisera USP2a Antisera USP2a Concentration Peptide 1 Peptide 2 1:500.79 0.87 1:250 0.58 0.58 1:1250 0.94 0.85 1:6250 0.93 0.63 1:31250 0.860.95 1:156000 0.64 0.53

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references topreferred embodiments thereof, it will be understood by those skilled inthe art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

What is claimed is:
 1. A method for diagnosing, staging or monitoringthe progression of a USP2a associated disease in a subject, the methodcomprising: a) obtaining a sample from the subject; b) contacting thesample with a nucleic acid molecule specifically hybridizable with anucleic acid molecule encoding a USP2a peptide, where the USP2a peptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO. 1, SEQ ID NO. 2 and variants thereof; c) determining a levelof USP2a in the sample detected by said nucleic acid moleculespecifically hybridizable with a nucleic acid molecule encoding a USP2apeptide; and d) comparing the level of the USP2a in the sample to abaseline level of the USP2a; wherein a difference in the level of USP2ain the sample as compared to the baseline level of greater than 1%overexpression is indicative of the presence, stage or progression ofthe USP2a associated disease in the subject.
 2. The method of claim 1wherein the nucleic acid molecule comprises RNA.
 3. The method of claim2 further comprising: e) modifying therapeutic intervention or treatmentof the USP2a disease or condition based on the difference in USP2alevels over or under the baseline.
 4. The method of claim 2 wherein thedetermination of the USP2a is by a method selected from the groupconsisting of polymerase chain reaction (PCR), reverse transcriptase-PCR(RT-PCR), in situ hybridization, Southern blot, Northern blot, sequenceanalysis, gene microarray analysis, and detection of a reporter gene. 5.The method of claim 1, wherein the subject is a patient.
 6. The methodof claim 5, wherein the patient has been diagnosed with a USP2aassociated disease.
 7. The method of claim 5, wherein the patient isundergoing treatment for a USP2a associated disease.
 8. The method ofclaim 1, wherein the baseline level of USP2a is the level of USP2a froman individual free of the USP2a associated disease.
 9. The method ofclaim 7, wherein the baseline level of USP2a is the level of USP2a fromsaid patient prior to any treatment.